The long-term objectives of this project have been to investigate the roles of viral DMA replication, replication proteins that serve as targets for antiviral drugs such as acyclovir, and gene expression in the interaction of herpes simplex virus (HSV) with the mammalian nervous system, especially virus latency. Latency is the most fascinating biological property of the virus and its most important clinical feature as it permits the virus to remain in the host and subsequently reactivate to cause disease. Understanding HSV latency is sure to reveal novel features of HSV and the nervous system. Such information is highly relevant to antiviral drugs that target DMA replication, vaccines, and potential agents to cure HSV infections. The proposed research will study the mechanisms by which acyclovir-resistant mutants retain pathogenicity, in particular the ability to reactivate from latency (aim 1). The relative contributions of low thymidine kinase (TK) expression and reversion will be assessed in experiments in which acyclovir will be administered to mice using implantable pumps during establishment of latency. Bacterial artificial chromosome methods will be used to map alleles of clinical isolates that compensate for loss of TK in reactivation. How viral gene expression is regulated during latency will be explored (aim 2). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays will be used to measure expression of different classes of transcripts during latency and reactivation. Knock-out mice will be used to test the hypothesis that immune effectors such as inteferon y repress the expression of viral genes. Why late transcripts accumulate during reactivation in the presence of acyclovir will be explored. A third set of studies will investigate whether the latency-associated transcript (LAT) locus represses viral gene expression through short intefering RNAs and microRNAs that can be expressed from this locus. The presence of these RNAs will be assayed in latently infected ganglia and, in collaboration with project 1, chromatin. Mutant viruses will be constructed to test the importance of these mechanisms for repression. Changes in host gene expression, which may affect latency, will also be investigated (aim 3). Array technologies, RT-PCR, and knockout mice will be used to identify whether observed changes in host gene expression are due to immune effectors such as interferon y. Viral mutants will be used to examine the role of viral factors (e.g. microRNAs) in these changes. The proposed experiments should shed light on viral and cellular factors involved in the establishment, maintenance, and reactivation of HSV latency. Latency is why herpesviruses cause diseases for which there are no cures. Understanding how latency is established, maintained, and reactivated could lead to a cure for HSV. Harvard Medical School, Boston, MA PHS 398(Rev. 09/04) Page Form Page 2 000135 Project 2 Principal Investigator/Program Director (Last, First, Middle): Schaffei", Priscilla A. KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Coen, Donald M. Harvard MedicalSchool PrincipalInvestigator Griffiths, Anthony Harvard Medical School Research Fellow Kramer, Martha F. Harvard MedicalSchool Research Associate Pesola, Jean Harvard MedicalSchool Research Fellow OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Moazed, Danesh Harvard Medical School Consultant Wang, Xiujie Chinese Academyof Sciences jCollaboratoij Human Embryonic Stem Cells E3 No Q Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://sterncells.nlh.gov/reaistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. Yes No PHS 398 (Rev. 09/04) Page Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. 000136 Project 2 Principal Investigator/ProgramDirector (Last, First, Middle): Schaffer, PriSCJIla Ann A. Specific Aims The long-term objectives of this project have been to investigate the roles of viral DNA replication and replication proteins (especially those that serve as targets for antiviral drugs), and gene expression in the interaction of herpes simplex virus (HSV) with the mammalian nervous system. These studies have implications for the biology of the virus and the nervous system, and for antiviral drugs and drug resistance. The goals are a subset of the overall goal of the program, which is to identify viral and cellular factors involved in the establishment/maintenance, and reactivation of HSV latency. The studies are very much in collaboration with the other members of the program. During the previous grant period, studies of thymidine kinase (tk) mutations that cause acyclovir-resistance (ACVr) in drug-resistant clinical isolates, have illuminated three different general ways by which the virus may evade drug therapy, yet retain pathogenicity. Studies detecting and quantifying HSV transcripts have raised the possibility of a specific block in late gene expression during latency, and revealed unexpected behaviors of late gene transcripts during reactivation. Other results from these studies and our recent detection of HSV- encoded microRNAs (miRNAs), have suggested mechanisms for how the latency-associated transcript (LAT) locus mediates repression during latency. Studies using array technology have revealed specific changes in neuronal gene expression in latently infected ganglia. These previous studies provide the basis for the current proposal whose specific aims are: 1. To study how ACV tk mutants from clinical isolates retain pathogenicity, focusing on the interplay of low level TK activity with reversion of frameshift mutations, and on alleles in genes other than tk that permit reactivation from latency in the absence of TK. 2. To investigate regulation of viral gene expression during HSV latency in mice, in particular to investigate regulation of late gene expression during latency and reactivation, and how repression may be mediated by the LAT locus and by immune effectors such as interferon (IFN) y. 3. To study changes in host gene expression during HSV latency in mice and how host and viral factors effect these changes. B. Background and Significance. The interaction of HSV with the mammalian nervous system that results in virus latency is the most fascinating biological property of the virus and its most important clinical feature. How does a virus that is so accomplished at productive infections completely alter its program of gene expression and replication to establish a silent, yet reactivatable infection of a neuron? Answering this question seems certain to uncover novel aspects of HSV and the nervous system. Such information is highly relevant to strategies to prevent and treat HSV infections and might even lead to agents to cure HSV infections. Moreover, studies of virus mutants with alterations in TK not only shed light on latency, but on mechanisms of gene expression and, because TK is a drug target, have implications for the use of antiviral drugs. Background information relevant to this proposal will now be reviewed: 1. HSV productive cycle ("lytic") gene expression. HSV is a large, enveloped virus containing a -150 kbp linear genome whose sequence predicts -70 protein coding genes (84). After entry of HSV DNA into the nucleus of cultured cells, a cascade of gene expression ensues [unless otherwise cited, see (97)]. Transcription of the first class of genes to be expressed (immediate early, IE, a) is stimulated by a virion component, VP16, and does not require prior viral protein synthesis. IE gene products include ICP 4, 27, 0, and 22, which are regulators of gene expression, and ICP47, which antagonizes antigen presentation. ICP4 and 27 are essential for viral replication. ICPO, ICP22, and ICP47 are non-essential. However, ICPO is especially important at low multiplicities of infection in resting cells and ICP22 is more important in some cell types than in others. The subsequent two classes of genes to be expressed are termed early (E, (3) and late (L, Y),respectively, based on when in the infectious cycle their expression peaks. E genes mainly encode proteins involved in DNA replication. These include enzymes that directly participate in DNA synthesis and are PHS 398/2590 (Rev. 09/04) Page Continuation Format Page 000137